Orai1 Mediates the Interaction between STIM1 and hTRPC1 and Regulates the Mode of Activation of hTRPC1-forming Ca2+ Channels*

Orai1 and hTRPC1 have been presented as essential components of store-operated channels mediating highly Ca2+ selective ICRAC and relatively Ca2+ selective ISOC, respectively. STIM1 has been proposed to communicate the Ca2+ content of the intracellular Ca2+ stores to the plasma membrane store-operated Ca2+ channels. Here we present evidence for the dynamic interaction between endogenously expressed Orai1 and both STIM1 and hTRPC1 regulated by depletion of the intracellular Ca2+ stores, using the pharmacological tools thapsigargin plus ionomycin, or by the physiological agonist thrombin, independently of extracellular Ca2+. In addition we report that Orai1 mediates the communication between STIM1 and hTRPC1, which is essential for the mode of activation of hTRPC1-forming Ca2+ permeable channels. Electrotransjection of cells with anti-Orai1 antibody, directed toward the C-terminal region that mediates the interaction with STIM1, and stabilization of an actin cortical barrier with jasplakinolide prevented the interaction between STIM1 and hTRPC1. Under these conditions hTRPC1 was no longer involved in store-operated calcium entry but in diacylglycerol-activated non-capacitative Ca2+ entry. These findings support the functional role of the STIM1-Orai1-hTRPC1 complex in the activation of store-operated Ca2+ entry.

Orai1 and hTRPC1 have been presented as essential components of store-operated channels mediating highly Ca 2؉ selective I CRAC and relatively Ca 2؉ selective I SOC , respectively. STIM1 has been proposed to communicate the Ca 2؉ content of the intracellular Ca 2؉ stores to the plasma membrane storeoperated Ca 2؉ channels. Here we present evidence for the dynamic interaction between endogenously expressed Orai1 and both STIM1 and hTRPC1 regulated by depletion of the intracellular Ca 2؉ stores, using the pharmacological tools thapsigargin plus ionomycin, or by the physiological agonist thrombin, independently of extracellular Ca 2؉ . In addition we report that Orai1 mediates the communication between STIM1 and hTRPC1, which is essential for the mode of activation of hTRPC1-forming Ca 2؉ permeable channels. Electrotransjection of cells with anti-Orai1 antibody, directed toward the C-terminal region that mediates the interaction with STIM1, and stabilization of an actin cortical barrier with jasplakinolide prevented the interaction between STIM1 and hTRPC1. Under these conditions hTRPC1 was no longer involved in store-operated calcium entry but in diacylglycerol-activated non-capacitative Ca 2؉ entry. These findings support the functional role of the STIM1-Orai1-hTRPC1 complex in the activation of store-operated Ca 2؉ entry.
Store-operated calcium entry (SOCE), 4 a process controlled by the filling state of the intracellular Ca 2ϩ stores (1), is a major mechanism for Ca 2ϩ influx in non-excitable cells. Because SOCE was first proposed two decades ago, many studies have been devoted to the identification of the mechanisms that communicate the Ca 2ϩ stores with the plasma membrane (PM) channels, as well as the nature of store-operated Ca 2ϩ (SOC) channels. The first identified and best-characterized store-operated current is I CRAC , but a number of other SOC currents activated by Ca 2ϩ store depletion have also been described (2).
The discovery of mammalian homologues of the Drosophila transient receptor potential (TRP) channel proteins has focused attention on TRP channels, especially the canonical TRP (TRPC) channels, as candidates for the conduction of SOCE (3)(4)(5) and a functional coupling between several TRPCs and IP 3 receptor isoforms (IP 3 Rs) has been demonstrated in transfected cells and cells naturally expressing TRPC proteins (4,6,7).
The recent identification of proteins STIM1 and Orai1 has shed new light on the nature and regulation of SOC channels. Orai1 (also named CRACM1 for Ca 2ϩ release-activated current (CRAC) modulator) has been proposed to form the pore of the channel mediating I CRAC (8). The involvement of Orai1 in I CRAC was identified by gene mapping in patients with hereditary severe combined immune deficiency syndrome attributed to loss of I CRAC (9,10). Orai1 has been demonstrated to form multimeric ion-channel complexes in the PM (11). The channel formed by Orai1 has been reported to be regulated by Ca 2ϩ store depletion with the participation of the intraluminal Ca 2ϩ sensor, stromal interaction molecule 1 (STIM1), a protein that has recently been presented as a messenger linking the endoplasmic reticulum (ER) to PM Ca 2ϩ channels. STIM1 is a Ca 2ϩbinding protein located mainly in the ER membrane with a single transmembrane region and a EF-hand domain in the NH 2 terminus located in the lumen of the ER (12), that might, therefore, function as a Ca 2ϩ sensor in the ER (13,14). Knockdown of STIM1 by RNA interference or functional knockdown of STIM1 by electrotransjection of neutralizing antibodies reduces SOCE in HEK293, HeLa, and Jurkat T cells and platelets (12,13,15) and I CRAC in Jurkat T cells (13). In support of the role of STIM1 in SOCE, mutation of the Ca 2ϩ binding EF-hand domain of STIM1 resulted in constitutive SOC channel activation without any detectable change in the content of the Ca 2ϩ stores (16). The cytoplasmic COOH-terminal domain of STIM1 has been suggested to interact with the NH 2 terminus of Orai1, facilitating the Orai1-STIM1 interactions required for the activation of I CRAC (17).
In addition, hTRPC1 has been presented as an essential component of the SOC channels. Heteromeric interactions of TRPC1 with other TRPCs have been reported to lead to the generation of SOC channels with different biophysical properties (18). In human platelets, hTRPC1 forms a complex hTRPC6, the type II IP 3 receptor and SERCA3 activated by depletion of the intracellular Ca 2ϩ stores (19). In addition, a recent study has reported that TRPC1 associates with STIM1 and Orai1 in culture cells to form a ternary complex that is important for the formation of the SOC channel (20). Orai1 has been shown to confer to TRPCs STIM1-mediated store-operated sensitivity (21); however, it remains unclear whether hTRPC1 interacts directly with STIM1 or through Orai1 and whether the heteromeric Orai1-TRPC1 interaction forms a channel sensitive to store depletion independently of STIM1.
In the present study we have investigated the interaction of endogenously expressed Orai1 with STIM1 and hTRPC1 at resting conditions and upon store depletion either by pharmacological tools or with the physiological agonist thrombin. In addition, we have investigated the role of the STIM1-Orai1 interaction on SOCE and the mode of activation of hTRPC1forming channels. Our results indicate that Ca 2ϩ store depletion stimulates rapid and transient interaction between Orai1 and both, STIM1 and hTRPC1. Electrotransjection with anti-Orai1 COOH terminus antibody or treatment with jasplakinolide (JP) prevented the interaction of STIM1 with Orai1 and hTRPC1, reduced SOCE, and changed the mode of activation of hTRPC1-forming channels.
Platelet Preparation-Platelet suspensions were prepared as previously described (22) as approved by Local Ethical Committees and in accordance with the Declaration of Helsinki. Briefly, blood was obtained from healthy drug-free volunteers and mixed with one-sixth volume of acid/citrate dextrose anticoagulant containing (in mM): 85 sodium citrate, 78 citric acid, and 111 D-glucose. Platelet-rich plasma was then prepared by centrifugation for 5 min at 700 ϫ g and aspirin (100 M) and apyrase (40 g/ml) were added. Platelets were then collected by centrifugation at 350 ϫ g for 20 min and resuspended in HEPES-buffered saline (HBS), pH 7.45, containing (in mM): 145 NaCl, 10 HEPES, 10 D-glucose, 5 KCl, 1 MgSO 4 and supplemented with 0.1% bovine serum albumin and 40 g/ml apyrase.
Cell viability was assessed using calcein and trypan blue. For calcein loading, platelets were incubated for 30 min with 5 M calcein-AM at 37°C, centrifuged, and the pellet was resus-pended in fresh HBS. Fluorescence was recorded from 2-ml aliquots using a Cary Eclipse spectrophotometer (Varian Ltd., Madrid, Spain). Samples were excited at 494 nm and the resulting fluorescence was measured at 535 nm. The results obtained with calcein were confirmed using the trypan blue exclusion technique. 95% of platelets were viable in our preparations.
Measurement of Intracellular Free Calcium Concentration ([Ca 2ϩ ] i )-Human platelets were loaded with fura-2 by incubation with 2 M fura-2/AM for 45 min at 37°C. Fluorescence was recorded from 2-ml aliquots of magnetically stirred cellular suspension (2 ϫ 10 8 platelets/ml) at 37°C using a Cary Eclipse spectrophotometer (Varian Ltd.) with excitation wavelengths of 340 and 380 nm and emission at 505 nm. Changes in [Ca 2ϩ ] i were monitored using the fura-2 340/380 fluorescence ratio and calibrated according to a established method (23).
Ca 2ϩ entry was estimated using the integral of the rise in [Ca 2ϩ ] i for 2.5 min after addition of CaCl 2 (22). OAG-induced Ca 2ϩ entry was estimated using the integral of the rise in [Ca 2ϩ ] i for 2.5 min after addition of OAG in a medium containing 1 mM Ca 2ϩ . Ca 2ϩ entry was corrected by subtraction of the [Ca 2ϩ ] i elevation due to leakage of the indicator or leak Ca 2ϩ entry after the addition of DMSO (the vehicle of TG and OAG). Ca 2ϩ release by TG was estimated using the integral of the rise in [Ca 2ϩ ] i for 3 min after the addition of the agent (22). Ca 2ϩ entry and release are expressed as nM⅐s, as previously described (24).
Immunoprecipitation and Western Blotting-The immunoprecipitation and Western blotting were performed as described previously (15). Briefly, 500-l aliquots of platelet suspension (2 ϫ 10 9 cell/ml) were lysed with an equal volume of RIPA buffer, pH 7.2, containing 316 mM NaCl, 20 mM Tris, 2 mM EGTA, 0.2% SDS, 2% sodium deoxycholate, 2% Triton X-100, 2 mM Na 3 VO 4 , 2 mM phenylmethylsulfonyl fluoride, 100 g/ml leupeptin, and 10 mM benzamidine. Aliquots of platelet lysates (1 ml) were immunoprecipitated by incubation with 2 g of anti-Orai1 antibody and 25 l of protein A-agarose overnight at 4°C on a rocking platform. The immunoprecipitates were resolved by 10% SDS-PAGE and separated proteins were electrophoretically transferred onto nitrocellulose membranes for subsequent probing. Blots were incubated overnight with 10% (w/v) bovine serum albumin in Tris-buffered saline with 0.1% Tween 20 (TBST) to block residual protein binding sites. Immunodetection of STIM1, hTRPC1, and Orai1 was achieved using the anti-STIM1 antibody diluted 1:250 in TBST for 2 h, the anti-hTRPC1 antibody diluted 1:200 in TBST for 1 h, and the anti-Orai1 antibody diluted 1:1000 in TBST for 1.5 h, respectively. The primary antibody was removed and blots were washed six times for 5 min each with TBST. To detect the primary antibody, blots were incubated for 45 min with horseradish peroxidase-conjugated ovine anti-mouse IgG antibody or horseradish peroxidase-conjugated donkey anti-rabbit IgG antibody diluted 1:10,000 in TBST and then exposed to enhanced chemiluminescence reagents for 4 min. Blots were then exposed to photographic films. The density of bands on the film was measured using scanning densitometry.
Reversible Electroporation Procedure-The platelet suspension was transferred to an electroporation chamber containing antibodies at a final concentration of 2 g/ml, and the antibod-ies were transjected according to published methods (15). Reversible electropermeabilization was performed at 4 kV/cm at a setting of 25-microfarad capacitance and was achieved by 7 pulses using a Bio-Rad Gene Pulser Xcell Electroporation System (Bio-Rad). Following electroporation, platelets were incubated with antibodies for an additional 60 min at 37°C and centrifuged at 350 ϫ g for 20 min and resuspended in HBS prior to the experiments.
Statistical Analysis-Analysis of statistical significance was performed using Student's t test. p Ͻ 0.05 was considered to be significant for a difference.

Orai1 Co-immunoprecipitates with hTRPC1 and STIM1 in Human
Platelets-Platelets have been shown to endogenously express hTRPC1 channel in the PM (5), and a functional interaction between STIM1 in the Ca 2ϩ stores and hTRPC1 has been reported to account for the activation of SOCE in these cells (15). We have now investigated the association between hTRPC1 and Orai1 by looking for co-immunoprecipitation from platelet lysates. Immunoprecipitation and subsequent SDS-PAGE and Western blotting were conducted using control platelets and platelets treated in the absence of extracellular Ca 2ϩ (100 M EGTA was added to the medium) for different periods of time (from 10 to 60 s) with inhibitor of the sarcoendoplasmic reticulum Ca 2ϩ -ATPase (SERCA) TG (1 M) plus a low concentration of ionomycin (50 nM), to induce extensive depletion of the intracellular stores in platelets (25). After immunoprecipitation with anti-hTRPC1 or anti-Orai1 antibodies, Western blotting revealed the presence of Orai1 in samples from resting platelets. The specificity of the hTRPC1 antibody was tested with the anti-TRPC1 antibody T1E3, which has been shown to be a specific tool in the investigation of mammalian TRPC1  proteins (5,26). We found that treatment with TG ϩ ionomycin increased the association between Orai1 and hTRPC1 in a time-dependent manner, reaching a maximal effect after 30 s of platelet stimulation (Fig. 1A, upper panel; n ϭ 6). Similar results were observed when cells were stimulated with the physiologi-cal agonist thrombin (1 units/ml) in a Ca 2ϩ -free medium (100 M EGTA was added at the time of experiment). Thrombin increased coimmunoprecipitation between Orai1 and hTRPC1 in a time-dependent manner, reaching a maximum after 10 s of stimulation with the agonist (Fig. 1B, upper panel; n ϭ 6). Western blotting of the same membranes with the antibody used for immunoprecipitation confirmed similar protein content in all lanes (Fig. 1,  lower panels).
Furthermore, we have explored the association between STIM1 and Orai1 by looking for co-immunoprecipitation from platelet lysates. Immunoprecipitation and subsequent SDS-PAGE and Western blotting were conducted using control platelets and platelets treated in a Ca 2ϩ -free medium (100 M EGTA added) for different periods of time (from 10 to 60 s) with TG (1 M) and ionomycin (50 nM). Our results indicate that treatment with TG ϩ ionomycin increased the association between Orai1 and STIM1 in a time-dependent manner, reaching a maximal effect after 10 s of platelet stimulation ( Fig. 2A, upper panel; n ϭ 6). Similar results were observed when cells were stimulated with thrombin, which increased co-immunoprecipitation between Orai1 and STIM1 in a timedependent manner, reaching a maximum after 30 s of stimulation with the agonist (Fig. 2B, upper panel; n ϭ 6). Western blotting of the same membranes with the antibody used for immunoprecipitation confirmed similar protein content in all lanes (Fig. 2, lower  panels). Our observations, showing an enhanced association of Orai1 with hTRPC1 and STIM1 in response to depletion of the intracellular Ca 2ϩ stores or the physiological agonist thrombin suggest that the STIM1-Orai1-hTRPC1 ternary complex might be important for the mediation of SOCE in these cells.
Inhibition of Store Depletion-evoked Interaction between STIM1 and hTRPC1 by Electrotransjection with Anti-Orai1 C-terminal Antibody-The amino acid sequence 288 -301 of human Orai1 recognized by the anti-Orai1 antibody used is FIGURE 3. Inhibition of store depletion-evoked interaction between STIM1 and hTRPC1 by electrotransjection with anti-Orai1 C-terminal antibody. A, human platelets were electropermeabilized in a Gene Pulser as described under "Experimental Procedures" and then incubated in the presence of 1 g/ml anti-Orai1 antibody (␣-Orai1) or 1 g/ml rabbit IgG (r-IgG) for 60 min as indicated. Cells were then stimulated with 1 M TG ϩ 50 nM ionomycin for 30 s and lysed. Whole cell lysates were immunoprecipitated (IP) in the absence of antibodies but adding protein A-agarose, and immunoprecipitated proteins were analyzed by Western blotting (WB) using anti-Orai1 antibody. These results are representative of four independent experiments. B, platelets (10 9 platelets/ml) were electropermeabilized or left untreated, as indicated, incubated for 60 min at 37°C in the absence of antibodies, and lysed. Whole cell lysates were subjected to Western blotting using anti-actin antibody as described under "Experimental Procedures." Positions of molecular mass markers are shown on the right. Histograms represent the quantification of actin in cells electropermeabilized and non-electropermeabilized. Results are presented as arbitrary optical density units and expressed as mean Ϯ S.E. of six independent experiments. C, human platelets (10 9 platelets/ml) were electropermeabilized and incubated with 1 g/ml rabbit IgG (r-IgG) or 1 g/ml anti-STIM1 antibody (␣-Orai1) for an additional 60 min at 37°C, as indicated. Cells were then incubated for 30 s in the absence or presence of 1 M TG ϩ 50 nM ionomycin in a Ca 2ϩ -free medium (100 M EGTA was added) and lysed. Whole cell lysates were immunoprecipitated with anti-STIM1 antibody. Immunoprecipitates were analyzed by Western blotting using anti-hTRPC1 antibody (upper panel) and reprobed with anti-STIM1 antibody (lower panel) as described under "Experimental Procedures." Positions of molecular mass markers are shown on the right. These results are representative of six independent experiments. located in the cytosolic COOH-terminal region of Orai1, which has been shown to be essential for the interaction of Orai1 with STIM1 (27). Since Orai1 has been proposed to mediate the communication between STIM1 and hTRPC1 (21) we have investigated whether the anti-Orai1 antibody, which is directed to the COOH-terminal region, could block the interaction between STIM1 and hTRPC1. To assess this possibility the anti-Orai1 antibody was introduced into platelets using an electropermeabilization technique. Electroporation can be used successfully for transferring antibodies into cells while maintaining the physiological integrity of the cells (15,28,29). Human platelets were reversibly electroporated as described under "Experimental Procedures." The presence of this antibody inside platelets was confirmed in samples from platelets electropermeabilized and incubated with 1 g/ml of either anti-Orai1 antibody or rabbit IgG, of the same nature of the anti-Orai1 antibody used, by immunoprecipitation without adding any additional antibody and subsequent Western blotting with the anti-Orai1 antibody. As shown in Fig. 3A, Orai1 was clearly detected in cells that had been previously electropermeabilized and incubated with anti-Orai1 antibody and not in cells incubated with rabbit IgG. Electropermeabilization allowed the anti-Orai1 antibody to enter the cells and immunoprecipitate native Orai1, which was then detected by Western blotting. To further investigate whether reversible electroporation might induce loss of proteins of the size of Orai1 (ϳ45 kDa) (30) we investigated the presence of actin (42 kDa) in electroporated and non-electroporated platelets. As shown in Fig. 3B, the amount of actin detected by Western blotting in electroporated platelets was not significantly smaller than that detected in non-electroporated platelets. Altogether, these findings confirm the efficacy of the electrotransjection and that the amount of Orai1 detected was not modified by treatment with TG ϩ ionomycin for 30 s (Fig. 3A, top panel).
As shown in Fig. 3C, interaction between STIM1 and hTRPC1 was abolished in platelets electrotransjected with 1 g/ml anti-Orai1 antibody (upper panel, third and fourth lanes; p Ͻ 0.001; n ϭ 6) compared with platelets electrotransjected with 1 g/ml rabbit IgG, as detected by immunoprecipitation of cell lysates with the anti-STIM1 antibody followed by Western blotting with anti-hTRPC1 antibody. Reprobing of the same membranes with anti-STIM1 antibody confirmed a similar protein loading in all lanes (Fig. 3C, lower panel). We found that electrotransjection of the anti-Orai1 antibody inhibits TG ϩ ionomycin-induced Orai1-STIM1 co-immunoprecipitation by performing immunoprecipitation with the transjected anti-Orai1 antibody (no additional antibodies were added for immunoprecipitation after transjection of anti-Orai1 antibody into cells) followed by Western blotting with the anti-STIM1 antibody (data not shown). These findings were not observed when platelets were electrotransjected with rabbit IgG (data not shown). These findings suggest that the amino acid sequence recognized by the anti-Orai1 antibody is essential for the interaction of STIM1 and hTRPC1, and blockade of this interaction might impair the function of the STIM1-Orai1-hTRPC1 ternary complex.

Impairment of the Interaction between STIM1 and hTRPC1 Changes the Behavior of hTRPC1-forming Channels from
Capacitative to Non-capacitative Channel-We have further investigated whether the anti-Orai1 antibody could affect SOCE in these cells. To assess this issue, the anti-Orai1 antibody was electrotransjected into platelets, followed by depletion of the intracellular Ca 2ϩ stores using TG (200 nM) to activate SOCE. Before the measurement of [Ca 2ϩ ] i platelets were maintained in a medium containing 200 M CaCl 2 , to avoid premature depletion of the stores. At the time of the experiment 250 M EGTA was added to perform the studies in a Ca 2ϩ -free medium. In platelets electrotransjected with rabbit IgG (Fig. 4A), TG evoked a prolonged elevation of [Ca 2ϩ ] i , due to leakage of Ca 2ϩ from intracellular stores (the integral for 3 min of the rise in [Ca 2ϩ ] i after the addition of TG was 238 Ϯ 73 nM⅐s; Fig. 4A, rabbit IgG: Control). Subsequent addition of Ca 2ϩ (1 mM) to the external medium induced a sustained increase in To assess the involvement of hTRPC1 in TGevoked SOCE we incubated cells for 30 min with 15 M anti-hTRPC1 antibody, directed toward the sequence 557-571 of human hTRPC1, which is located in the pore-forming region between the fifth transmembrane domain and region VII of hTRPC1 (31). We have previously used this procedure to successfully block hTRPC1 channel function (5,32). Incubation with the anti-hTRPC1 antibody significantly reduced TG-evoked SOCE without having any effect on TG-induced Ca 2ϩ release (TG-induced Ca 2ϩ release and entry, estimated as the integral of the rise in [Ca 2ϩ ] i after the addition of TG or CaCl 2 were 252 Ϯ 51 and 441 Ϯ 72 nM⅐s, respectively; Fig. 4A, rabbit IgG:␣-hTRPC1; p Ͻ 0.05). These findings confirm the role of hTRPC1 in SOCE. In platelets electrotransjected with anti-Orai1 antibody and not incubated with anti-hTRPC1 antibody TG-induced Ca 2ϩ entry was significantly reduced compared with cells electrotransjected with rabbit IgG (the integrals of the rise in [Ca 2ϩ ] i after the addition of TG or CaCl 2 were 212 Ϯ 27 and 297 Ϯ 34 nM⅐s, respectively; Fig. 4B, ␣-Orai1: Control; p Ͻ 0.05). Interestingly, incubation with anti-hTRPC1 antibody did not significantly modify either Ca 2ϩ release or entry induced by TG in platelets electrotransjected with anti-Orai1 antibody (the integrals of the rise in [Ca 2ϩ ] i after the addition of TG or CaCl 2 were 204 Ϯ 23 and 343 Ϯ 58 nM⅐s, respectively; Fig. 4B, ␣-Orai1:␣-hTRPC1). As mentioned under "Experimental Procedures," the integrals were corrected by subtraction of the elevation in [Ca 2ϩ ] i observed after the addition of 1 mM Ca 2ϩ in cells treated with vehicle (DMSO) instead of TG ( Fig. 4; leak Ca 2ϩ entry). These findings indicate that hTRPC1 is not involved in SOCE in platelets electroporated with anti-Orai1 antibody. Because some Ca 2ϩ entry was still detectable in platelets electrotransjected with anti-Orai1 antibody, our results indicate that a TRPC1-Orai1-independent pathway is involved in the remaining Ca 2ϩ entry in these cells.
To investigate whether hTRPC1 is involved in non-capacitative Ca 2ϩ entry in platelets electrotransjected with anti-Orai1 antibody we used OAG, a diacylglycerol (DAG) analogue that induces non-capacitative Ca 2ϩ entry in human platelets (32,33). In the absence of extracellular Ca 2ϩ OAG was unable to induce elevation in [Ca 2ϩ ] i (data not shown). In platelets electrotransjected with rabbit IgG, OAG (100 M)-induced Ca 2ϩ entry was not significantly modified by incubation for 30 min with 15 M anti-hTRPC1 antibody (the integral of the rise in [Ca 2ϩ ] i for 2.5 min after the addition of OAG was 1509 Ϯ 194 and 1479 Ϯ 193 nM⅐s in platelets incubated in the absence or presence of anti-hTRPC1 antibody, respectively; Fig. 5A; n ϭ 6). In platelets electrotransjected with anti-Orai1 antibody, treatment with OAG enhances non-capacitative Ca 2ϩ entry to 137% of control (rabbit IgG: Control versus ␣-Orai1:Control). Incubation for 30 min with 15 M anti-hTRPC1 antibody reduced OAG-mediated non-capacitative Ca 2ϩ entry to 89% of control (rabbit IgG:Control versus ␣-Orai1: ␣-hTRPC1; the integral of the rise in [Ca 2ϩ ] i for 2.5 min after the addition of OAG was 2073 Ϯ 252 and 1348 Ϯ 236 nM⅐s in platelets incubated in the absence and presence of anti-hTRPC1 antibody, respectively; Fig. 5, B and C; n ϭ 6). These findings suggest that impairment of the interaction between hTRPC1 and STIM1 results in a change in behavior of hTRPC1, or heteromeric channels including hTRPC1, from capacitative to non-capacitative Ca 2ϩ entry.
Finally, we have investigated the functional relevance of the interaction between STIM1 and hTRPC1 by testing the effect of JP, a cell-permeant peptide isolated from Jaspis johnstoni, which induces polymerization and stabilization of actin filaments (34). JP has been shown to elongate and organize actin filaments exclusively at the cell periphery and we have previously used it to stabilize the membrane actin cytoskeleton in platelets and prevent the interaction between ER and PM (35). Treatment of human platelets with 10 M JP for 30 min at 37°C resulted in a significant inhibition of the interaction between hTRPC1 and STIM1 as detected by co-immunoprecipitation (Fig. 6A). In addition, in the presence of JP, non-capacitative Ca 2ϩ entry stimulated by 100 M OAG was enhanced, an effect that was reduced by incubation with anti-hTRPC1 antibody to 64% (Fig. 6, B versus C; p Ͻ 0.05; n ϭ 6), reaching a value that was comparable with that induced by OAG in the absence of JP (Fig. 6C, JP: ␣-hTRPC1 versus Fig. 6B, Control).

DISCUSSION
Canonical TRP proteins have been shown to form both storeoperated (capacitative) and non-capacitative Ca 2ϩ permeable channels. The latter has been reported to be gated downstream of phospholipase C (PLC) activation by the second messenger DAG, or its analog OAG (36 -38), or by other lipid messengers, such as lysophosphatidic acid (39) and sphingosine 1-phosphate (40). hTRPC1 has long been proposed as a candidate to mediate SOCE by a dynamic interplay with the ER Ca 2ϩ sensor STIM1 and the PM channel Orai1 (20). STIM1 has been reported to act as a regulator of different store-operated Ca 2ϩ currents mediated, among others, by Orai-and TRPC1-forming channels (43). Although Orai1 has been presented as the channel mediating the Ca 2ϩ selective I CRAC , whereas TRPC1 has been shown to participate in the conduction of other rela-tively Ca 2ϩ selective I SOC (2), a functional requirement for Orai1 in the generation of TRPC1-SOC channels has recently been proposed (44). Here we show that impairment of the interaction between STIM1 and Orai1 results in inhibition of the interaction between STIM1 and hTRPC1, which indicates that Orai1 mediates the communication between STIM1 and hTRPC1. In addition, we have observed that impairment of the interaction between STIM1 and hTRPC1 results in loss of storeoperated (capacitative) behavior of hTRPC1-forming channels and appearance of DAG-regulated noncapacitative behavior. To our knowledge, this is the first description of this change in behavior of hTRPC1, because hTRPC1 has never been shown to respond to OAG. Other TRPC channel proteins have been reported to function in two distinct ways depending on the expression level or even the mode of expression. In the B lymphocyte cell line DT40 an increase in the level of expression of TRPC3 results in the disappearance of store-operated behavior and the appearance of a receptor-activated non-capacitative behavior (41). In addition, TRPC7 has been shown to be activated by both receptor-and store-operated modes in HEK-293 cells depending on the mode of expression. When stably expressed, TRPC7 can be activated by either Ca 2ϩ store depletion or PLC activation; however, when transiently expressed TRPC7 forms channels activated downstream of PLC, but not by Ca 2ϩ store depletion (42). Human platelets naturally express different hTRPC proteins, including hTRPC1, hTRPC3, hTRPC4, hTRPC5, and hTRPC6 (45,46). Thus, the enhanced OAG-mediated Ca 2ϩ entry observed when the interaction between STIM1 and Orai1-hTRPC1 is impaired might not be due to homo-hTRPC1 tetramers, but to a heteromeric channel with hTRPC1 as one of the subunits. We believe that the change in behavior of Ca 2ϩ -permeable channel involving hTRPC1 in human platelets is unlikely mediated by an increase in the level of expression or by a different mode of expression because we are investigating endogenously expressed hTRPC1 and presumably the duration of the experiments (1 h preincubation with anti-Orai1 antibody) is not long enough to induce any change in protein expression in anucleated platelets.
We have tested the functional relevance of Orai1, mediating the interaction between STIM1 and hTRPC1, on the mode of activation of the hTRPC1-forming channel by electrotransjection of cells with an anti-Orai1 antibody, directed toward the COOH-terminal region, which is involved in the interaction with STIM1 (27). We have further tested the role of STIM1 in the mode of activation of hTRPC1-forming channels by treatment with JP. JP induces the formation of a cortical actin barrier at the PM, so excluding cytoplasmic organelles from this region and thus preventing close association between the PM and internal organelles (47). We have found that JP causes elongation and reorganization of actin filaments exclusively near the PM and reduces SOCE in platelets and other cells (35,48,49). A model based on the interaction between STIM1 in the ER and Orai1-hTRPC1 complex in the PM might be expected to be affected by stabilization of the cortical cytoskeleton barrier by JP before store depletion. Both electrotransjection with the anti-Orai1 antibody and treatment with JP prevented the interaction between STIM1 and hTRPC1, the former suggesting that this interaction is mediated by Orai1. In addition, both experimental maneuvers reduced SOCE (see Ref. 35 for JP) and enhanced OAG-mediated noncapacitative Ca 2ϩ entry. In platelets with impaired interaction between STIM1 and Orai1-hTRPC1, incubation with the anti-hTRPC1 antibody had no effect on the remaining SOCE but reduced OAG-evoked non-capacitative Ca 2ϩ entry to a level that was found to be similar to OAG-mediated non-capacitative Ca 2ϩ entry in cells where the STIM1-Orai1-hTRPC1 interaction was allowed, thus suggesting that under these conditions channels involving hTRPC1 support non-capacitative Ca 2ϩ entry. Our findings are in agreement with previous studies by Birnbaumer's group (21,50) reporting that SOCE/I CRAC channels are composed of heteromeric complexes including TRPCs and Orai proteins, with Orai conferring STIM1-mediated store depletion sensitivity to these channels.
Furthermore, we present for the first time evidence for the interaction between endogenously expressed Orai1 and hTRPC1 and between Orai1 and STIM1 in platelets. The interaction between these proteins was detected in resting platelets and enhanced by store depletion or by the physiological agonist thrombin reaching a maximum after 10 or 30 s of stimulation and then decreased. The interaction between Orai1 and hTRPC1 or STIM1 was found to be independent on extracellular Ca 2ϩ . We have previously demonstrated that STIM1 co-immunoprecipitates with hTRPC1, which is likely involved in the communication of the Ca 2ϩ content of the ER to hTRPC1-forming channels to mediate SOCE (15). These findings further support the involvement of a dynamic STIM1-Orai1-hTRPC1 ternary complex in the activation of SOCE as previously reported in cultured cells (20).
Our findings suggest that Orai1 mediates the communication between STIM1 and hTRPC1, which is essential for the mode of activation of channels including hTRPC1 as a subunit. We propose that under normal conditions STIM1 in the ER interacts with the complex Orai1-hTRPC1 in the PM and induces the activation of the hTRPC1-forming channel by store FIGURE 7. Speculative model for the regulation of hTRPC1 channel behavior by STIM1 in platelets. Top, in cells with a functional interaction between STIM1, Orai1, and hTRPC1, occupation of membrane receptors by an agonist results in the activation of PLC through a G-protein, leading to the synthesis of IP 3 and DAG. The latter induces non-capacitative Ca 2ϩ entry (NCCE) and IP 3 activates IP 3 receptors (IP 3 R) in the ER, induces Ca 2ϩ release and activates store-operated (capacitative) Ca 2ϩ entry (CCE) through hTRPC1 and other plasma membrane channels. Bottom, impairment of the interaction between STIM1 and Orai1 by electrotransjection of an anti-Orai1 (C-terminal) antibody, inhibits the interaction between STIM1 and hTRPC1 leading to a change in behavior of hTRPC1-forming channels from capacitative (store-operated) Ca 2ϩ channel to DAG-activated noncapacitative Ca 2ϩ channel. depletion. In contrast, when the communication between STIM1 and the Orai1-hTRPC1 complex is prevented hTRPC1forming channels support non-capacitative Ca 2ϩ entry, perhaps by forming heteromeric channels with other hTRPC subunits activated by OAG (a schematic diagram of the proposed model is depicted in Fig. 7). These data support that STIM1 regulates hTRPC1 activation mode.